Study On Inhibition Of Bovine Viral Diarrhea Virus By ShRNA

Bovine viral diarrhea virus (BVDV) is the pathogen of BVD-MD (bovine viral diarrhea mucosal disease). The disease is associated with reduction of milk yield, lowered breeding success, respiratory disease and higher susceptibility to other infections. It occurs worldwide and has considerable economic consequences. Currently control of infection is by slaughter or active immunisation, but neither are very effective.BVDV is a single-stranded RNA virus, the RNA containing a single open-reading frame (ORF) encoding for a large poly-protein. Proteolytic cleavage of the protein produces a range of structural and non-structural viral proteins. In the mammalian host the presence of the single stranded RNA elicits an innate immune response and the production of class1interferon, leading in turn to a non-specific translational shutdown and apoptosis.RNA interference (RNAi) is a mechanism involves the processing of double stranded RNA duplexes in to small interfering RNA (siRNA). These combine with complementary sequences in mRNA and effectively silence a gene’s expression. Direct transfection of cells with in vitro DNA-based vectors, along with RNA polymerase(polⅢ) promoter, expressing short hairpin RNA (shRNA) molecules that are processed within the cell to produce active siRNA molecules can be used to block gene expression.We have investigated the use of such a protocol to target BVDV expression. Two shRNA recombinant expression vectors, pSGH1-1083and pSGHl-8235, targeted to the conserved sequence of the BVDV genome were constructed. They were annealed and ligated into the BglⅡ and HindⅢ site of linearized pSGHl vector. The recombinant expression vectors were identified by PCR, restriction enzyme digestion and sequencing. The successfully constructed vectors, pSGH1-sh1083and pSGH1-sh8235along with a control vector not containing the shRNA components, were tested for their effectiveness in suppressing BVDV expression in mammalian cells. MDBK cells were transfected and cells showing high stable expression were cloned and tested for suppression following infection with BVDV. Cells expressing pSGHl-sh1083or pSGHl-sh8235showed a reduced cytopathic effect at72h, electrophoresis showed no expression of the BVDV E2gene and the percentage of cells showing BVDV by flow cytometry analysis was reduced.This study demonstrated successfully that shRNA-carrying vectors specific for BVDV genes will disrupt viral expression and growth in mammalian cells. This now provides a practical basis for the further investigation of the molecular determinants of pathogenicity in BVDV.